A 42 kb region on human chromosome 9p21 encodes for three distinct tumor suppressors, p16INK4A, p14ARF and p15INK4B, and is altered in an estimated 30–40% of human tumors. The expression of the INK4A-ARF-INK4B gene cluster is silenced by polycomb during normal cell growth and is activated by oncogenic insults and during aging. How the polycomb is recruited to repress this gene cluster is unclear. Here, we show that expression of oncogenic Ras, which stimulates the expression of p15INK4B and p16INK4A, but not p14ARF, inhibits the expression of ANRIL (antisense non-coding RNA in the INK4 locus), a 3.8 kb-long non-coding RNA expressed in the opposite direction from INK4A-ARF-INK4B. We show that the p15INK4B locus is bound by SUZ12, a component of polycomb repression complex 2 (PRC2), and is H3K27-trimethylated. Notably, depletion of ANRIL disrupts the SUZ12 binding to the p15INK4B locus, increases the expression of p15INK4B, but not p16INK4A or p14ARF, and inhibits cellular proliferation. Finally, RNA immunoprecipitation demonstrates that ANRIL binds to SUZ12 in vivo. Collectively, these results suggest a model in which ANRIL binds to and recruits PRC2 to repress the expression of p15INK4B locus.

The ubiquitin–proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F‐box protein and substrate recognition component of an Skp1–Cullin–F‐box protein (SCF) ubiquitin ligase, were generated. Although Skp2−/− animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis. Skp2−/− cells also exhibit increased accumulation of both cyclin E and p27Kip1. The elimination of cyclin E during S and G2 phases is impaired in Skp2−/− cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27Kip1 is mediated by the SCFSkp2 ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators.

Overexpression of transforming growth factor ␤ (TGF-␤) has been shown to play pathogenic roles in progression of renal fibrosis, and the severity of tubulointerstitial fibrosis correlates better with renal function than the severity of glomerulosclerosis. Smad proteins are signaling transducers downstream from TGF-␤ receptors. Three families of Smad proteins have been identified: receptorregulated Smad2 and Smad3, common partner Smad4, and inhibitory Smad7 (part of a negative-feedback loop). We investigated Smad-mediated TGF-␤ signaling pathway and regulatory mechanisms of inhibitory Smad7 in unilateral ureteral obstruction (UUO) kidneys in mice, a model of progressive tubulointerstitial fibrosis. Compared with sham-operated kidneys, the level of Smad7 protein, but not mRNA, decreased progressively in UUO kidneys, whereas immunoreactivity for nuclear phosphorylated Smad2 and Smad3 and renal fibrosis were inversely increased. Furthermore, we demonstrated that both the degradation and ubiquitination activity of Smad7 protein were increased markedly in UUO kidneys compared with sham-operated ones. We also found that both Smurf1 and Smurf2 (Smad ubiquitination regulatory factors), which are E3 ubiquitin ligases for Smad7, were increased and that they interacted with Smad7 in UUO kidneys. Our results suggest that the reduction of Smad7 protein resulting from enhanced ubiquitin-dependent degradation plays a pathogenic role in progression of tubulointerstitial fibrosis.transforming growth factor ␤ ͉ Smad proteins ͉ tubulointerstitial fibrosis

Many types of human papillomavirus (HPV) are associated with genital lesions. In order to develop simple and sensitive diagnostic procedures for HPV infection, we took advantage of the polymerase chain reaction (PCR). We compared the published nucleotide sequences of the LI region from six genital HPV types and designed a pair of consensus primers for LI region. The PCR with the consensus primers for LI region (Ll‐PCR) could amplify at least nine genital HPV types, 6,11, 16, 18, 31, 33, 42, 52 and 58, and the amplified HPV DNA could be typed by subsequent restriction mapping. Ll‐PCR was compared to Southern blot analysis and also to the consensus primer‐mediated PCR for E6 region (E6‐PCR) described before. Although both our PCR systems are nonradioactive, the sensitivity in detecting HPV DNA was even better than that obtained by using Southern blot analysis. By means of the PCR systems we detected HPV DNA in 100% of cervical condylomas (10/10), 92% of cervical intraepithelial neoplasias (33/36) and 96% of invasive cervical carcinomas (53/55), while we detected HPV DNA in 12% of normal cervices (12/102).

The identity of the cellular mechanisms through which nitroglycerin (glyceryl trinitrate, GTN) elicits nitric oxide (NO)-based signaling to dilate blood vessels remains one of the longest standing foci of investigation and sources of controversy in cardiovascular biology. Recent evidence suggests an unexpected role for mitochondria. We show here that bioconversion by mitochondria of clinically relevant concentrations of GTN results in activation of guanylate cyclase, production of cGMP, vasodilation in vitro, and lowered blood pressure in vivo, which are eliminated by genetic deletion of the mitochondrial aldehyde dehydrogenase (mtALDH). In contrast, generation of vasoactivity from alternative nitro(so)-vasodilators is unaffected. In mtALDH ؊/؊ mice and their isolated vascular tissue, GTN bioactivity can still be generated, but only at substantially higher concentrations of GTN and by a mechanism that does not exhibit tolerance. Thus, mtALDH is necessary and sufficient for vasoactivity derived from therapeutic levels of GTN, and, more generally, mitochondria can serve as a source of NObased cellular signals that may originate independently of NO synthase activity.nitric oxide ͉ nitrite ͉ S-nitrosothiol ͉ nitrate tolerance T he ability of mammalian cells to convert the manmade organic nitrate, nitroglycerin (glyceryl trinitrate, GTN), to vasoactive nitric oxide (NO) or S-nitrosothiol (SNO) played a significant part in the discovery that NO or its equivalent functions as an endogenous physiological mediator (1, 2), and GTN has long served as a principal therapeutic agent for acute angina and congestive heart disease (3-7). Although multiple cellular activities mediating GTN metabolism have been characterized, the mechanisms that specifically subserve GTN bioactivation have remained elusive. Recent evidence indicates a central role for mitochondria in GTN bioactivation. In particular, it has been proposed that the mitochondrial aldehyde dehydrogenase (mtALDH), aldehyde dehydrogenase (ALDH) 2, may provide a principal enzymatic source of GTN-derived NO vasoactivity (through the intermediacy of mitochondrial nitrite) and that mechanism-based mtALDH inactivation contributes to GTN tolerance (8-10). However, this previously uncharacterized role for mitochondria in the generation of NO bioactivity remains unproven, and the centrality of mtALDH in GTN bioactivation in vivo has been disputed (5-7). The findings described in the present study establish that NO bioactivity originating in mitochondria and generated by mtALDH is necessary and sufficient to account for the vasoactivity of clinically relevant concentrations of GTN. Our results suggest, in addition, that inactivation of mtALDH is a principal component of mechanism-based GTN tolerance. Materials and Methods mtALDH ؊/؊ Mice. The generation of mtALDHϪ/Ϫ mice has been described in ref. 11. Wild-type (C57BL͞6) and mtALDH Ϫ/Ϫ mice used for comparative measurements were gender-and agematched (3-4 months). All procedures were approved by the Institutional Animal Care and...